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Abstract

Introduction

The objective of this study was to investigate the effects of tumor necrosis factor
(TNF)-α inhibitors on circulating T helper-type 17 (Th17) cells and Th17-related cytokines
in patients with rheumatoid arthritis (RA).

Methods

The frequencies of circulating Th17 cells and serum levels of Th17-related cytokines
were determined using flow cytometry analysis and ELISA, respectively, in 48 RA patients
both before (baseline) and six months after anti-TNF-α therapy. Therapeutic response
was evaluated using European League Against Rheumatism (EULAR) response criteria.

Conclusions

The beneficial effect of anti-TNF-α therapy might involve a decrease in Th17-related
cytokines in responders, whereas rising levels of circulating Th17-cells and IL-17
were observed in patients with an inadequate response to anti-TNF-α therapy.

Introduction

Rheumatoid arthritis (RA) is characterized by the infiltration of macrophages and
T cells into the joints, synovial hyperplasia, cartilage degradation and bone erosions
[1]. Tumor necrosis factor (TNF)-α is a crucial inflammatory mediator in rheumatoid synovitis
and subsequent tissue damage in RA [2,3]. Although TNF-α inhibitors can be an effective and well-tolerated therapy for RA
patients [4-6], a significant proportion of patients do not acquire advantageous effects [7]. In addition, the effect of TNF-α inhibitors on the immune response has not been
fully explored.

T helper-type 17 (Th17) cells, a novel and distinct subset of Th cell, can secrete
interleukin (IL)-17 in humans [8-10]. Interleukin-17 is a pleiotropic cytokine that participates in tissue inflammation
and destruction by inducing the expression of pro-inflammatory cytokines and matrix
metalloproteases [8,11,12]. The frequencies of Th17 cells were found to increase in peripheral blood mononuclear
cells (PBMCs) of RA patients compared to healthy controls [13,14]. An enhanced expression of IL-17 has been observed in the rheumatoid synovium [15] and synovial fluids of patients with early RA [16]. Interleukin-17 in vitro stimulates the production of TNF-α and IL-1β, and also synergizes with TNF-α to induce
cartilage loss and promote osteoclastogenesis [17,18]. A recent study showed that Th17 cells, but not Th1 cells, cooperate with synovial
fibroblasts in a pro-inflammatory feedback loop that drives chronic destruction in
RA [19]. Moreover, IL-17 has become a new therapeutic target for animal models with collagen-induced
arthritis (CIA) and human RA [20-22]. These observations suggest that Th17 cells and IL-17 critically contribute to synovitis
and bone destruction associated with RA.

Recently, TNF-α was shown in vitro to drive the production of IL-17 with the ability to differentiate T cells towards
a Th17 phenotype [23]. In a psoriasis-like skin inflammation model, TNF-α enhanced the expression of Th17-related
cytokine genes during priming but suppressed these cytokine transcripts when present
during re-stimulation [24]. In CIA, TNF-α inhibitors reduced the number of Th17 cells in pathologic joints despite
an increase of Th17 cells in inguinal lymph nodes [25]. Taken together, these findings show that TNF-α blockade has paradoxical effects
on the expression of Th17-related cytokines in animal models of autoimmune diseases.

In humans, an engineered p75 TNFRII dimer, etanercept, suppressed the gene expression
levels of Th17-related cytokines including IL-6 and IL-23 in cutaneous lesions of
psoriasis [26]. Kageyama et al. also reported a significant decrease in serum levels of IL-23 at three and six months
after etanercept therapy in RA patients [27]. TNF-α inhibitor, adalmumab, reduced the frequency of circulating Th17 cells and
serum IL-6 levels in RA patients [28]. However, a recent study showed that an increased frequency of circulating Th17 cells
after TNF-α blockade is accompanied by a decrease in Th17-specific chemokine receptor
expression in RA [29]. When taken together, these results reveal conflicting effects of TNF-α inhibitors
on Th17 cells and IL-17 in humans.

In the present study, we attempted to determine whether or not the clinical response
to anti-TNF-α therapy of RA patients led to changes in the levels of circulating Th17
cells and Th17-related cytokines, and we also investigated their clinical implication.

Materials and methods

Patients

A total of 48 consecutive patients (39 females and 9 males; mean age ± SD 50.1 ± 13.5
years), who fulfilled the 1987 revised criteria of the American College of Rheumatology
for RA [30], were evaluated before and six months after anti-TNF-α therapy. All patients remained
in active disease in spite of treatment with methotrexate (MTX) and other disease-modifying
anti-rheumatic drugs (DMARDs), for whom anti-TNF-α therapy was initiated based on
the British Society for Rheumatology guidelines [31]. Fourteen patients received etanercept at a dose of 25 mg twice weekly and 34 patients
received adalimumab at a dose of 40 mg every other week in combination with a stable
dose of MTX of 7.5 to 15 mg weekly. Corticosteroids (≦10 mg/day) and non-steroid anti-inflammatory
drugs (NSAIDs) were allowed but were given at stable doses for at least four weeks
before and during the six-month anti-TNF-α therapy. Disease activity was assessed
by the 28-joint disease activity score (DAS28) [32]. The therapeutic response was evaluated six months after anti-TNF-α therapy was started,
according to the European League Against Rheumatism (EULAR) response criteria [33]. The patients were categorized into good, moderate or non-responders based on the
amount of change in the DAS28 and the level of DAS28 reached. Good responders are
defined as patients who have a decrease in DAS28 from baseline (ΔDAS28) of >1.2 and
a DAS28 at the sixth month of < 3.2; moderate responders have either ΔDAS28 of >1.2
and a DAS28 at the sixth month of ≧3.2 or ΔDAS28 of 0.6 to 1.2 and a DAS28 at the
sixth month of < 5.1; and non-responders are those who have either ΔDAS28 of < 0.6
or a DAS28 at the sixth month of ≧5.1 [33]. To obtain a better analysis, we combined good responders and moderate responders
into EULAR responders. Twelve age- and sex-matched healthy volunteers (10 females
and 2 males, 47.6 ± 8.1 years), who had no rheumatic disease, were used as normal
controls. Blood samples were collected at baseline (before starting anti-TNF-α therapy)
and six months after anti-TNF-α therapy. The Ethics Committee of Taichung Veterans
General Hospital approved this study and the written consent of each participant was
obtained.

Quantitation of circulating Th17cells using flow cytometry analysis

In order to detect circulating Th17 cells, phycoerythrin (PE)-conjugated anti-IL-17
(eBioscience, San Diego, CA, USA) and Phycoerythrin-Cyanin 5 (PC5)-conjugated anti-CD4
(Beckman Coulter, Marseilles, France) were quantified using flow cytometry according
to the manufacturer's protocol and a technique previously described [34,35]. Briefly, aliquots of 1,000 μl of the sterile heparinized whole blood were stimulated
with a combination of 25 ng/ml of phorbol myristate acetate and 1 μg/ml of ionomycin
(Sigma, Deisenhofen, Germany) and cultured for one hour at 37°C in a humidified 5%
CO2 incubator. Whole blood was treated with 10 μg/ml of Brefeldin A (Sigma, Germany) to
inhibit intracellular protein transport. Activated cultures of blood samples were
washed in wash buffer (phosphate buffered saline, 5% foetal bovine serum, 0.1% sodium
azide; Merck, Darmstadt, Germany) and then stained with 20 μl of PC5-conjugated CD4-specific
monoclonal antibody (mAb) (Beckman Coulter, Marseilles, France) for 15 minutes at
room temperature (RT). Erythrocytes were lysed by adding 2 ml of fluorescence-activated
cell sorter (FACS) lysing solution (Becton Dickinson, Lincoln Park, NJ, USA). After
five minutes of incubation, the samples were centrifuged and washed with 0.1% BSA-PBS,
and subsequently fixed with 100 μl Reagent 1 (Beckman Coulter, Marseilles, France)
for 10 minutes. After washing, the pellet was incubated with 100 μl Reagent 2, saponin
(Beckman Coulter, Marseilles, France) for five minutes at RT in the dark. The samples
were washed twice with 0.1% BSA-PBS and then incubated with PE-conjugated IL-17-specific
mAb (eBiosciences, San Diego, CA, USA) for 30 minutes at RT in the dark. An isotype
control IgG1-PE (eBiosciences, USA) was used for the IL-17 staining at RT in the dark.
After staining, the cells were washed and immediately analysed using flow cytometry
(Beckman Coulter, USA). Lymphocytes were gated on the basis of forward- and side-
scatter properties and at least 10,000 CD4+ cells were analysed. The results were analysed using Expo32 software (Beckman Coulter,
Miami, FL, USA).

Determination of serum levels of Th17-related cytokines by ELISA

Serum levels of IL-6, IL-17, IL-21, IL-23 and TNF-α were determined in 48 RA patients
at baseline and after six months of anti-TNF-α therapy, and in 12 healthy controls
using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions
(eBiosciences, USA).

Determination of the anti-CCP antibody was performed by ELISA using a commercial kit
(INOVA Diagnostics Inc., San Diego, CA, USA). A result was considered positive for
anti-CCP antibodies if the titer was above 20 IU/ml. Serum levels of RF-IgM were measured
by nephelometry (Dade Behring Inc., Newark, DE, USA). A result was considered positive
for RF when the concentration was above 15 IU/ml.

Statistical analysis

The results are presented as the mean ± SD or median (interquartile range, IQR). The
non-parametric Mann-Whitney U test was used for between-group comparisons of serum
levels of IL-6, IL-17, IL-21, IL-23 and TNF-α, and for the percentages of circulating
Th17 cells. The correlation coefficient was obtained by the non-parametric Spearman's
rank correlation test. The Wilcoxon signed rank test was used to compare the percentages
of circulating Th17 cells and serum levels of Th17-related cytokines during follow-up
for the RA patients after anti-TNF-α therapy. A probability of less than 0.05 was
considered significant.

Results

Baseline characteristics of RA patients with a different therapeutic response

As illustrated in Table 1, the majority of RA patients were female and all patients had active disease (DAS28,
mean ± SD, 7.22 ± 0.79) before starting anti-TNF-α therapy. After anti-TNF-α therapy,
36 (75.0%) patients were EULAR responders, including 20 (41.7%) good responders and
16 (33.3%) moderate responders, whereas 12 (25.0%) were non-responders. Although non-responders
had higher erythrocyte sedimentation rates (ESR) and RF titers compared to responders,
the differences did not reach statistical significance. There were also no significant
differences in the baseline demographic data, positive rate of RF and anti-CCP antibodies,
daily dose of corticosteroids, or the proportion of patients previously using DMARDs
between responders and non-responders.

As shown in Figure 1, the baseline serum levels of Th17-related cytokines including IL-17, IL-6, IL-21
and IL-23, were significantly higher in RA patients than in healthy controls. Among
the RA patients, significantly higher IL-17 levels were observed in non-responders
than in responders (P < 0.01, Figure 2 and Table 2). Although non-responders seemed to have higher levels of IL-6 and TNF-α when compared
to the levels in responders, the differences did not reach statistical significance.
There was also no significant difference in the levels of Th17-related cytokines between
the etanercept-treated group and the adalimumab-treated group (data not shown).

Table 2. Changes in levels of Th17-cells and Th17-related cytokines in RA patients with different
therapeutic responses

Correlation between the frequencies of circulating Th17 cells and disease activity
or serum levels of Th17-related cytokines in RA patients

Multiple logistic regression analysis showed that only a high baseline IL-17 level
(≧40.0 pg/ml) could significantly predict a poor response to anti-TNF-α therapy (P < 0.01), with a medium level of specificity (83.3%) and sensitivity (66.7%).

Discussion

Consistent with the findings of previous reports [36,37], our results showed that 36 (75.0%) patients have moderate or good EULAR response
to the six-month anti-TNF-α therapy and 12 (25.0%) were non-responders. Although non-responders
seem to have higher baseline ESR, titers of RF and anti-CCP antibodies, and serum
levels of TNF-α and IL-6, when compared to responders, the differences do not reach
statistical significance. Our findings suggest that an inadequate response to anti-TNF-α
therapy is not merely a reflection of high disease activity.

The present study is the first attempt to investigate the effects of TNF-α inhibitors
on the levels of circulating Th17 cells and Th17-related cytokines in RA patients
with a different therapeutic response. In order to obtain a better reflection of in vivo cytokine patterns than is achievable with PBMCs, whole blood was stimulated with mitogens
and lymphocytes double-stained with IL-17 and CD4 were analysed using flow cytometry.
Our results showed significantly higher baseline frequencies of circulating Th17 cells
in active RA patients compared with healthy controls, confirming the findings of previous
studies [13,14,29]. We also showed that the baseline levels of circulating Th17 cells were positively
correlated with DAS28 scores in RA patients. Our data and the findings of previous
studies [15,16] suggest a potential role of Th17 cells in the pathogenesis of RA.

Similar to the results of recent studies [28], our findings showed a significant decrease in the frequencies of circulating Th17
cells after anti-TNF-α therapy, in parallel with the decrease in DAS28 in EULAR responders.
Although the diseases studied were different, our results were also consistent with
the findings of previous studies which showed that the TNF-α inhibitor reduced Th17
cell responses with the amelioration of psoriatic skin lesions [38] and that the TNFR1 inhibitor suppressed the Th17 response with clinical benefits
in experimental autoimmune encephalomyelitis mice [39]. In contrast, our results showed a significant increase in circulating Th17 cell
frequencies in non-responders. Our findings in non-responders were similar to the
results of a recent study that showed an increased frequency of circulating Th17 cells
after TNF-α blockade in RA patients [29]. The divergent effects on Th17 cells in vivo after anti-TNF-α therapy for RA patients suggest that there may be different subsets
of the immune response to TNF-α inhibitors. It is tempting to speculate that the change
in frequencies of circulating Th17 cells may help to differentiate between EULAR responders
and non-responders because these changes occur within the first six months of anti-TNF-α
therapy.

The Th17 cells have a specific role in immune function through the production of effector
cytokines. Through the secretion of IL-17, Th17 cells act on the differentiation of
osteoclasts and bone resorption [40], and can stimulate the monocytes to produce pro-inflammatory cytokines, thus amplifying
the inflammatory cascade [11,17,18]. In the present study, we showed that serum IL-17 levels were significantly elevated
and correlated with DAS28 in active RA patients. Our data were consistent with the
results of recent studies describing an increase in IL-17 levels in RA patients [14,41], and they also supported the results of clinical trials showing the therapeutic benefits
of IL-17 blockade [20-22]. During a longitudinal follow-up of RA patients who received anti-TNF-α therapy,
we found that serum IL-17 levels significantly decreased, in parallel with the clinical
remission in the responders, whereas increasing IL-17 levels were found in non-responders.
Whether or not this imbalance of Th17 cells and IL-17 between PB and affected joints
contributes to the change in Th17-related cytokines after anti-TNF-α therapy remains
unclear.

Accumulating evidence indicates that IL-6 can enhance Th17 cell differentiation by
promoting the sequential engagement of IL-21/IL-23 pathways and that it plays a critical
role in Th17-dependent autoimmune diseases [42]. In the present study, we showed that the high baseline levels of IL-6 were positively
correlated with the frequencies of circulating Th17 cells in RA patients. After anti-TNF-α
therapy, a significant decrease in the levels of circulating Th17 cells and IL-17,
in parallel with the decrease in DAS28 and serum IL-6 levels was observed in responders.
These observations support the role of IL-6 in the differentiation of Th17 cells [42-44], and the therapeutic benefits of IL-6 receptor inhibitors in RA patients [45].

Interleukin-21 is required to reinforce differentiation of Th17 cells, and it plays
a critical role in Th17-dependent autoimmune diseases [46]. In contrast to IL-12, IL-23 does not promote the development of Th1 cells, but it
is crucial for the expansion and maintenance of Th17 cells [47]. In the present study, we showed that the baseline levels of IL-21 and IL-23 were
significantly elevated in active RA patients, supporting their role in the pathogenesis
of this disease [42,48]. In addition, we found that serum levels of IL-21 and IL-23 significantly decreased,
in parallel with the clinical remission in responders after anti-TNF-α therapy. Consistent
with our data, the results of the study by Kageyama et al. also showed a significant decrease in serum IL-23 levels at three and six months after
anti-TNF-α (etanercept) therapy in RA patients [27].

The mechanisms of the therapeutic effects of TNF-α inhibitors on RA have not been
fully explored. Lundy et al. showed that TNF-α has a positive feedback effect on Th17 cells, causing a vicious
cycle of synovitis [49]. TNF-α also promotes IL-17 production by inducing dendritic cells to direct the differentiation
of Th cells towards the Th17 phenotype [23], and by the administration of TNF-like ligand 1A enhancing Th17 differentiation with
IL-17 production [50]. Our results showed a significant decrease in the levels of circulating Th17 cells
and TNF-α after effective therapy with TNF-α inhibitors, suggesting that the down-regulation
of both Th17-related cytokines and TNF-α may be one of the mechanisms of anti-TNF-α
therapy [19]. Our findings support the hypothesis that anti-TNF-α therapy could reduce Th17 responses
by blocking the TNF-α-dependent positive feedback system [23]. Another explanation may be the inhibition of emigration of Th17 cells from lymphoid
organs after anti-TNF-α therapy [24].

In the present study, we showed that one-fourth of RA patients have an inadequate
response to anti-TNF-α therapy despite a significant decrease in serum TNF-α level,
suggesting that neutralization of TNF-α cannot fully explain the therapeutic effect
of TNF-α inhibitors in non-responders. Additionally, significantly higher baseline
levels of circulating Th17-cells and IL-17 were observed in non-responders when compared
to responders. During a longitudinal follow-up of RA patients undergoing anti-TNF-α
therapy, we found a significant increase in circulating levels of Th17-cells and IL-17
in non-responders. Although the cut-off value for baseline IL-17 (40 pg/mL) used in
our study was not absolute, a multivariate logistic regression analysis showed that
only a high baseline level of IL-17 could be a significant predictor of poor therapeutic
response to TNF-α inhibitors. Our observations suggest that an inadequate response
to anti-TNF-α therapy may reflect TNF-independent but Th17-dominant inflammatory process,
which has been observed in previous studies [15,16].

This was a preliminary study that enrolled a limited number of active RA patients
who were followed up for six months. Because we mainly investigate the effects of
anti-TNF-α therapy on Th17 cells and Th17-related cytokines, we did not include a
group of RA patients without anti-TNF-α therapy as a disease control. Because the
patients enrolled in our study were not an early RA population, our results might
not be directly applicable to early RA patients undergoing anti-TNF-α therapy. Therefore,
a long-term study enrolling a larger group of patients, including an additional early
RA population, or a control group using DMARDs only, is required to validate these
findings. Although the erroneous increase in IL-17 levels may have been due to the
heterophilic binding by serum RF [51,52], there were no significant differences in RF positivity or titers between responders
and non-responders in the present study.

Conclusions

Our results show that anti-TNF-α therapy not only neutralizes the effects of TNF-α,
but it also down-regulates the IL-6/Th17 axis in EULAR responders. In contrast, anti-TNF-α
therapy causes a significant increase in Th17 cells in RA patients with an inadequate
response to anti-TNF-α therapy. A high baseline level of IL-17 may have a predictive
value for poor therapeutic response to TNF-α inhibitors. Although the small sample
size of this study did not allow for a definitive conclusion, our findings at least
suggest that Th17-dependent inflammation is implicated in the pathogenesis of a subset
of RA patients with poor response to TNF-α inhibitors, and that treatment targeting
IL-17 may be beneficial for these patients [21,22].

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

DYC and JLL participated in the study design, acquisition of data, interpretation
of results, and manuscript preparation. YMC participated in the study design, acquisition
of data, interpretation of results, and assisted in drafting the manuscript. HHC,
CWH and CCL contributed to analysis and interpretation of data, and assisted in drafting
the manuscript. All authors approved the final manuscript.

Acknowledgements

This study was supported by grant TCVGH-993803C from Taichung Veterans General Hospital,
Taiwan.

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